The long-range project objectives are to understand how folding information is encoded in the sequence of membrane proteins and the means by which the cell recognizes failure of this process. Studies of the cystic fibrosis transmembrane conductance regulator (CFTR) supported by this grant have provided fundamental information relevant to understanding the structure of the nucleotide binding (NBD) and transmembrane (TMD) domains, the kinetic and thermodynamic effects of cystic fibrosis (CF)-causing folding mutants, and the quality control mechanisms that assist folding and recognize misfolding CFTR molecules. Future studies build on this foundation by detailing the folding pathway of the NBD and the final assembly of CFTR domains with each other and with domains of other proteins at the membrane. To this end the four specific aims are to: 1. Characterize the folding pathway of NBD1 and determine the effects of the deltaF508 and other CFmutations. Building on the high resolution structure of CFTR-NBD1, biophysical and biochemical methods will be employed to characterize the pathway(s) of folding and the effects of nucleotide and CF-causing mutations on the pathway. 2. Assess the ability of NBD1 and NBD2 to form dimers and determine the effect of CF-causing mutations on these interactions. Nucleotide binding domains of ABC transporter homologues of the CFTR channel form ATP-dependent dimers, with two nucleotides sandwiched at two active ATPase sites formed at the dimer interface. We will test the hypothesis that CFTR NBD1 and NBD2 form an ATP sandwich dimer with a single active ATPase site and an inactive "regulatory" site. 3. Assess the ability of the NBDs and the transmembrane domains (TMDs) to interact and determine the effect of CF-causing mutations on these interactions. The backbone at the F508 position is critical for NBD1 folding, but the side chain is important for the later folding steps of domain association. We will test the hypothesis that F508 lies at the TMD/NBD1 interface. 4. Assess the ability of the STAS domain of SLC26A3 to bind CFTR and determine the effect of this on CFTR domain-domain association. To understand the coordinated regulation of CFTR channel and apical SLC26A anion exchange activity, we will elucidate the molecular mechanism by which the STAS domain of SLC26A3 activates CFTR. To accomplish these four goals, a combination of biochemical, biophysical, and cell biological approaches will be employed. These studies are necessary for and fundamental to a detailed understanding of the mechanisms by which membrane proteins fold.